The Gram staining method is one of the widely used differential staining methods in bacteriology, established by the Danish doctor, Hans Christian Gram, in 1884. The bacteria first were stained by the basic primary stain, followed by the addition of a mordant (Gram's iodine), and then decolorized with ethyl alcohol. Some of the bacteria remain their stain color without decolorization in certain conditions, while some are decolorized. Therefore the bacteria can be classified into two groups, the former is Gram-positive (G+) bacteria and the latter is Gram-negative (G−) bacteria. For clear observation, a final step of counter-staining with safranin or basic fuchsin is applied after decolorization. The color of Gram-positive remains purple while the Gram-negative bacteria were stained to appear red. The Gram-positive bacteria include Bacillus, most of the coccus such as Staphylococcus, Streptococcus, Enterococcus, and all of the actinobacteria and the eubacteria. The Gram-negative bacteria include Vibrio, Spirochaeta and Bacteria.
The Gram-positive bacteria and the Gram-negative bacteria turn up differently on their chemical, physical properties and their staining results as well. Currently, it is generally acknowledged that the Gram-positive bacteria have unique complexes of nucleoprotein magnesium salt and polysaccharide, and the combinations between the complexes of the Gram-positive bacteria and the complexes of crystal violet and iodine (CV-I) within the inner and outer layers of the cell are strong, therefore the Gram-positive bacteria are not easy to be decolorized. In the contrast, the combinations between the complexes of the Gram-negative bacteria and the complexes of crystal violet and iodine (CV-I) within the inner and outer layers of the cell are not so strong, it causes that the Gram-negative bacteria can not absorb stains efficiently so that they can be decolorized easily. Generally, the staining mechanism is based on above principle.
Besides, the isoelectric point of the Gram-positive bacteria is much lower than the isoelectric point of the Gram-negative bacteria, so the Gram-positive bacteria can absorb many stains and be much difficult to be decolorized, while the Gram-negative bacteria are opposite under the same PH condition. Therefore, the staining conditions need to be controlled strictly. For example, in the strong base condition to progress staining, both types of the Gram-positive bacteria and the Gram-negative bacteria can absorb basic stains, therefore, it results in the positive reaction. While under the low PH condition, both of them appear the negative result. Moreover, the penetrations between the crystal violet-iodine complex and the cell walls of these two types of bacteria are not the same. The Gram-positive bacteria have lower penetration so that they are not easy to be decolorized. The Gram-negative bacteria have higher penetration so they can be easily decolorized. Therefore, the time and method for decoloriztion should be controlled strictly.
Gram Staining Mechanism:
Gram-positive bacteria have thick mesh-like cell walls made of peptidoglycan to form penetration barriers. During decolorization by the ethyl alcohol, the thicker Gram-positive cell walls dehydrate so as to close the pores as the cell walls shrink. As a result, the crystal violet-iodine complexes are blocked on the cell walls, and the bacteria remain stained and appear purple. In contrast, the Gram-negative bacteria are those bacteria having thinner layer of peptidoglycan, much loose linkage. Using ethyl alcohol to decolorize can not shirk the structure of bacteria anymore. Also, the Gram-negative bacteria contain a much higher amount of lipids, and ethyl alcohol can dissolve the lipids and increase the pores so the crystal violet-iodine complexes can be removed from the cell walls. After, a counterstain (commonly safranin) colors all Gram-negative bacteria a red color.
The Gram stain method generally includes four basic steps of applying a primary stain (crystal violet), a mordant (Gram's iodine), decolorization, and counter-staining. The specific operation includes the following steps of:
1) applying the bacteria to a slide and affixing the slide;
2) staining the bacteria with ammonium oxalate crystal violet for 1 min;
3) rinsing with water;
4) treating the slide with the mordant solvent and then waiting for 1 min;
5) rinsing the slide with water and using water-absorbing paper to absorb the extra moisture;
6) adding drops of 95% ethyl alcohol and slightly shaking to progress decolorizing for 30 seconds, rinsing with water, drying it; and
7) counter-staining the slide with safranin for 10 seconds, rinsing with water, drying it and examining with a microscope.
The result appears that Gram-positive cells remain purple while the Gram-negative cells turn to red.
The four-steps Gram staining method has great result but the operation steps are much more complicated. Owing to the busy lab working, each technique is required to be much simpler and more efficient in order to accomplish more tasks in such limited time.
In view of the drawbacks of the prior art, the inventor of the present invention based on years of experience conducted extensive researches and experiments, and finally developed a two-step gram staining method to overcome the drawbacks of the prior art.